The vitamin K epoxide reductase complex (VKORC) recycles the reduced form of vitamin K which is an essential cofactor for post-translational γ-carboxylation of vitamin K dependent (VKD) proteins (Nelsestuen et al. (1974) The mode of action of vitamin K. Identification of gamma-carboxyglutamic acid as a component of prothrombin. J. Biol. Chem., 249, 6347-6350). The VKORC1 gene was identified recently, and is described in detail in Rost et al. (2004) Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature, 427, 537-541).
VKD proteins contain γ-carboxylated glutamate (gla) residues giving them specific biochemical and physiological properties like Ca-dependent binding to negatively charged phospholipid membranes in the case of blood clotting factors (Mann et al. (1988) Cofactor proteins in the assembly and expression of bloodclotting enzyme complexes. Annu. Rev. Biochem., 57, 915-956). VKD proteins include procoagulant factors II, VII, IX and X, and anticoagulant proteins C, S and Z. Although restricted to one single known enzymatic reaction, γ-carboxylase activity is found in all mammalian tissues (Vermeer and de Boer-van den Berg MA (1985) Vitamin K-dependent carboxylase. Haematologia (Budap.), 18, 71-97). The γ-carboxylase catalyzes a carboxylation reaction using reduced vitamin K as cofactor.
Vitamin K dependent (VKD) gamma carboxylation of glutamic acid residues is a post-translational protein modification required for the generation of biologically active VKD proteins playing roles in hemostasis, growth control, calcium homeostasis, and signal transduction (Furie et al. (1999) Vitamin K-dependent biosynthesis of gamma-carboxyglutamic acid. Blood, 93, 1798-1808; Berkner, K. L. (2000) The vitamin K-dependent carboxylase. J. Nutr., 130, 1877-1880). Several glutamic acid residues in the N-terminal Gla-domain of these proteins are modified by carboxylation to enable calcium-dependent phospholipid membrane interactions (Stenflo and Suttie (1977) Vitamin K-dependent formation of gamma-carboxyglutamic acid. Annu. Rev. Biochem., 46, 157-172; Suttie (1980) Mechanism of action of vitamin K: synthesis of gamma-carboxyglutamic acid CRC Crit Rev. Biochem., 8, 191-223). These multiple gamma-glutamate (Gla) residues allow the Gla domain to undergo conformational changes which are required for the activity of VKD proteins in combination with binding to phospholipid membrane surfaces (Nelsestuen et al. (1976) Role of gamma-carboxyglutamic acid. Cation specificity of prothrombin and factor X-phospholipid binding. J. Biol. Chem., 251, 6886-6893; Zwaal et al. (1998) Lipid-protein interactions in Blood coagulation. Biochim. Biophys. Acta, 1376, 433-453).
The VKD blood coagulation proteins require full or nearly full carboxylation to bind to membrane surfaces in the presence of calcium ions (Furie and Furie (1988) The molecular basis of blood coagulation. Cell, 53, 505-518). If vitamin K antagonists inhibit gamma carboxylation, thus undercarboxylated VKD proteins cannot form the calcium dependent structure which results in low affinity to phospholipids membranes and less activity (Esmon et al. (1975a) A new carboxylation reaction. The vitamin K-dependent incorporation of H-14-CO3-into prothrombin. J. Biol. Chem., 250, 4744-4748; Esmon et al. (1975b) The functional significance of vitamin K action. Difference in phospholipid binding between normal and abnormal prothrombin. J. Biol. Chem., 250, 4095-4099; Malhotra, O. P., Nesheim, M. E., & Mann, K. G. (1985) The kinetics of activation of normal and gamma-carboxyglutamic acid-deficient prothrombins. J. Biol. Chem., 260, 279-287). For example, contributions to overall protein activity losses could be assigned to the absence of each of the 10 Gla-residues of the VKD protein activated human protein C (Zhang et al. (1992) Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity. Blood, 80, 942-952). Missing procoagulant activity of undercarboxylated factor IX mutants found in hemophilia B patients can be assigned to impaired calcium-induced conformational changes and loss in the ability to bind phospholipid vesicles (Ware et al. (1989) Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties. J. Biol. Chem., 264, 11401-11406).
In case of recombinant factor IX, it has been shown that expression of functional factor IX in Chinese hamster ovary cells is limited by the fact that carboxylation ability is saturated at higher production levels (Kaufinan et al. (1986) Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. J. Biol. Chem., 261, 9622-9628; Derian et al. (1989) Inhibitors of 2-ketoglutarate-dependent dioxygenases block aspartyl beta-hydroxylation of recombinant human factor IX in several mammalian expression systems. J. Biol. Chem., 264, 6615-6618).
Recombinant over-expression of γ-carboxylated proteins was shown in case of human factor IX to lead to a limitation of propeptide cleavage and γ-carboxylation at higher secretion rates, thus yielding proteins which are only partially occupied with gla residues also when vitamin K is available in the culture medium in surplus. This leads to the secretion of variants of VKD recombinant proteins with reduced activities. Addition of vitamin K to the medium did not improve factor IX activity at high expression levels. The requirement of vitamin K present in the cell culture medium to elicit active factor IX was shown to reach saturation at 5 μg/ml. Below this level, the secreted amount of active factor IX from Chinese hamster ovary (CHO) cells was dependent on vitamin K concentration (Kaufinan et al. (1986) Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. J. Biol. Chem., 261, 9622-9628).
Up to now cell lines with low expression levels have to be chosen for production in order to overcome these limitations of cellular capacity to modify VKD proteins post-translationally. Co-expression of Furin, the propeptide cleaving enzyme, leads to complete cleavage of this propeptide (Wasley et al. (1993) PACE/furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway. J. Biol. Chem., 268, 8458-8465), but is not involved in γ-carboxylation improvement. Another approach, the overexpressing of γ-carboxylase, has not led to improved protein secretion in case of factor IX (Rehemtulla et al. (1993) In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. U.S.A, 90, 4611-4615). Factor IX molecules, which are bound to the carboxylase during the carboxylation reaction are not released effectively. It was concluded that the supply of reduced vitamin K form at the site of γ-carboxylation is the limiting step of this reaction (Hallgren et al. (2002) Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: implications for the release of vitamin K-dependent proteins. Biochemistry, 41, 15045-15055).
Therefore, a strong need exists for stabilizing the expression, particularly the recombinant expression of VKD proteins in host organisms yielding in improved secretion rates and/or activities of the expressed VKD proteins.
Thus, it is an object of the present invention to provide new systems and methods for improving the productivity of (particularly recombinant) VKD protein expression via co-expression of VKORC1.